The focus of this program is the development of the immune system, using the South African clawed frog, Xenopus laevis, as a model. The advantages of using Xenopus laevis as a model are that amphibian embryos develop externally, and that as a consequence there is no interference of the maternal environment during embryonic development. Past work has involved a characterization of the first immunoglobulin synthesized during development, and an investigation of several other properties of the early immune system. In addition, a cDNA encoding part of an IgM heavy chain was cloned and used to measure the onset of immunoglobulin synthesis during development. The cDNA probe was also used to identify a clone from a reticulocyte genomic lambda library that had cross-hybridizing sequences. Subsequent analysis revealed that this clone contained a Mu constant region pseudogene. During the period of this application, we propose to characterize the structure and sequence of heavy chain genes from both cells which do not synthesize immunoglobulin and those that do. In this way, we will analyze the contribution of gene rearrangement to the generation of antibody diversity in these amphibians. We will also examine the structure of immunoglobulin mRNA during development with particular reference to the time of appearance of the mRNA for secreted and membrane-bound immunoglobulin forms. In summary, we will continue to study the molecular changes involved in the development of the immune system at a stage where these events are difficult or impossible to study in mammals as they occur in utero.